BCL11A Facilitates Cell Proliferation and Metastasis in Neuroblastoma via Regulating the PI3K/Akt Signaling Pathway

Purpose: The study aims to access the value of B-cell lymphoma/leukemia 11A (BCL11A) in the prognosis of patients with neuroblastoma (NB) and to explore its role and possible mechanism in NB. Methods: Tumor specimens from 53 children with neuroblastoma were evaluated for the relationship between BCL11A expression level and prognosis of NB patients. Online datasets like SEQC and Asgharzadeh were analyzed to further check out the suppose.The role of BCL11A in the proliferation and migration of NB cells was studied by functional experiments such as CCK8, colony formation, flow cytometry, transwell and wound healing assay after knocking down BCL11A by small interfering RNA (siRNA) in vitro. The protein makers of the potential pathways were tested by western blot. Results: High expression of BCL11A in NB patients was closely correlated with high-risk and poor prognosis. The proliferation and migration abilities of NB cell lines SK-N-BE(2) and IMR-32 were significantly impaired by silencing BCL11A. Downregulation of BCL11A expression level in NB cells inhibited the epithelial-mesenchymal transition (EMT) process and affected the PI3K/Akt signaling pathway. Conclusion: As a prognostic indicator of survival in NB patients, BCL11A might serve as a potential therapeutic target. BCL11A played a regulatory role in cell proliferation, invasion, and migration in NB, which may be through the PI3K/AKT signaling pathway and induce EMT.


INTRODUCTION
Neuroblastoma (NB) is the most common extracranial solid tumor in early childhood, accounting for approximately 8% to 10% of all pediatric tumors and 15% of tumor-related deaths in children [1]. NB is an enigmatic tumor with high heterogeneity in clinical outcomes, with spontaneous regression among low-risk cases but relentless progression in highrisk ones. With the progress in the therapeutic area in the recent two decades, the survival of low-and intermediaterisk NB are improved substantially. However, the long-term survival rate among patients with high-risk NB is lower than 50% [2,3]. Therefore, new therapeutic strategies for children with high-risk NB are urgently needed.
*Address correspondence to this author at the Department of Pediatric Hematology/Oncology, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China; Tel: +86-21-25078360; E-mail: yuanxiaojun@xinhuamed.com.cn # This author contributed equally to this work.
As a transcription factor, B-cell lymphoma/leukemia 11A (BCL11A) plays an essential role in normal development, such as the switch of hemoglobin, migration of projection neurons, and lymphopoiesis [4]. BCL11A was initially recognized in B-cell non-Hodgkin lymphoma (B-NHL) and Hodgkin's disease (HD) [5]. Recently, BCL11A has attracted widespread attention as a prognostic marker and potential therapeutic target in malignant neoplasms. The aberrant expression of BCL11A has been reported in acute myeloid leukemia [6], natural killer/T-cell lymphoma [7], breast cancer [8], lung cancer [9], and so on. Its increased expression resulted in malignant proliferation and metastasis, induced epithelial-mesenchymal transition and anticancer drug resistance, and its high expression level is associated with poor prognosis [10].
Moreover, the expression level of BCL11A is significantly elevated in high-risk neuroblastoma [11]. As a target of miR-146a, BCL11A can promote SK-N-SH cell growth and inhibit apoptosis [12]. However, the biological functions and clinical significance of BCL11A in NB remain unexplored.
This study aimed to investigate the relationship between BCL11A expression level and the clinical features in NB patients, as well as to explore the potential effect of BCL11A as a prognostic marker for NB patients. What's more, the biological role of BCL11A in neuroblastoma was checked by silencing the BCL11A expression in neuroblastoma cell lines to explore its mechanism of tumor progression in NB.

Clinical Samples and Cell Culture
Patients diagnosed with neuroblastoma in Xin Hua Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, in the period of Sep. 2016 to July 2019, were enrolled to collect clinical information and tumor samples for further investigation. All patients were treated according to the Chinese Children Cancer Group-NB-2014 (CCCG-NB-2014) protocol [13]. This study was approved by the Ethics Committee of Xin Hua Hospital affiliated with Shanghai Jiao Tong University School of Medicine. The cell lines SK-N-SH, IMR-32, SH-SY5Y, and 293T were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). SK-N-BE(2) and SK-N-AS were purchased from ATCC (Manassas, USA). SK-N-SH, IMR-32, SK-N-BE(2), and 293T were all cultured in DMEM supplemented with 10% fetal bovine serum(FBS) and 1% penicillin-streptomycin solution. SH-SY5Y and SK-N-AS cell lines were cultured in a 1:1 mixture of MEM and F12 Medium with 1% Gluta-max, 1% Sodium pyruvate, 1% NEAA, 1% penicillin-streptomycin solution, and 10% FBS. The mediums and FBS were all purchased from Gibco, USA. All cells grew in a humidified incubator with 5% CO2 at 37°C.

BCL11A Expression Analysis in Human Cancer and NB Cell Lines
The BCL11A mRNA levels in different human cancers were analyzed by the Tumor Immune Estimation Resource (TIMER) database. The PrognoScan database (http://www. prognoscan.org/) was used to study the relationship between BCL11A expression and the prognosis of cancer patients. BCL11A expressions in normal tissues, tumors, and cell line datasets were visualized with the R2 genomics analysis and visualization platform (http://r2.amc.nl/). Additionally, the NB datasets SEQC and Asgharzadeh with information on clinical and prognostic factors were also available at the R2 genomics analysis and visualization platform.

Immunohistochemical (IHC) Staining and Semi-quantitative Analysis
After dewaxed, rehydrated, and processed for antigen retrieval, the paraffin-embedded sections were quenched with 3% H 2 O 2 and blocked with 3% BSA. The slide was incubated with the primary antibody anti-BCL11A (1:100, Abcam) and with an HRP-conjugated secondary antibody. Staining was visualized by incubation with DAB.
Stained slides were evaluated by 2 experienced pathologists. Based on the staining intensity and the positive rate, a semiquantitative integration method was used to determine the results. The staining intensity score was as follows: colorless = 0, light yellow = 1, yellow-brown = 2, and brown = 3. The percentage of positive cells was scored as follows: < 25% = 1, 26% -50% = 2, 51% -75%= 3, >75% = 4. The result of staining was determined using the following formula: score = percentage score × staining intensity score [14,15]. Median expression of BCL11A was used as the cutoff and patients were divided into high (n=27) and low (n=26) expression groups.

Cell Transfection
Neuroblastoma cells were transfected by the Lipofectinmediated transfection method. The siRNA specific for BCL11A was synthesized (RiboBio) and the sequence was GGCAGCCTCTGCTTAGAAA. The control sequence was CAACAGGAGAGACCTTTAT. Cells were plated in sixwell plates 24 h prior to transfection. According to the manufacturer's instructions (www.thermofisher.cn/), the cell lines were cultured with the transfection complex (concluded 200ul Opti-MEM I, 6ul siRNA, 8ul Lipofectamine RNAiMAX, and complete medium) when the cell density reached 30-40%. After 24 h, the medium was changed to a complete medium. When cells reached 70-80% confluency, siRNA expressing cells were selected using a growth medium with 2 μg/mL puromycin (Beyotime). The Lipofectamine RNAiMAX was purchased from Invitrogen and Opti-MEM I medium was from Gibco.

Cell Proliferation Assay
Cell proliferation assay was performed at 48h after transfection, including Cell Counting Kit-8 (CCK-8; Yeasen), colony formation assay, and flow cytometry. For CCK-8, control and si-BCL11A NB cells were seeded on 96-well plates with 5 × 10 3 cells per well. And each group was set in five parallel holes. The cells were incubated with 10μL CCK8 solution and 90μl RPMI 1640 phenol-free red medium in each well. The optical density was measured after 2 hours. Cell proliferation was examined at 0, 24, 48, and 72 hours.
For colony formation assay, cells were seeded on 6-well plates (1.0 ×10 3 cells per well) and cultured for approximately 10 days. After the formation of colonies, cells were stained with crystal violet and then counted through five randomly selected fields by microscope at ×40 magnification, under bright-field illumination.
Cell cycle distribution was determined by flow cytometry. Cells in the logarithmic growth phase were collected and fixed with 70% ethanol overnight at 4°C. On the next day, the centrifuged cells were stained with 500μl propidium iodide (PI) Triton X-100 solution in the dark for 30 minutes at 37 o C. All experiments were performed with at least 3 replicates.

Cell Invasion and Migration Analysis
Tumor cell migration ability was assessed by wound healing assay. The cell lines were transfected with siRNA and incubated in 6-well plates until cell density reached 95%. The confluent monolayer of cells was scratched by a sterile pipette tip and then incubated with serum-free DMEM and observed at ×200 magnification for 5 specific visual fields at 0h and 24h of wound initiation. The scratch healing rate =scratch width at (0h -24h) / 0h × 100%.
Invasion ability was analyzed through a transwell assay. Transwell assay was conducted after 48 hours of transfection with siRNA. The upper surfaces of the polycarbonic membranes of the transwell chambers (Corning Costar) were coated with 1:6 DMEM diluted Matrigel (Invitrogen). The lower chambers were filled with 500μL of DMEM supplemented with 15% FBS. Cells (2×10 5 ) in 200μL DMEM were seeded into the upper chambers. After 48h incubation, cells that had migrated on the lower surface of the filters were fixed in 4% paraformaldehyde and stained with crystal violet. The cells were counted in 5 randomly selected visual fields under the microscope at ×200 magnification. All experiments were also performed with at least 3 replicates.

Statistical Analysis
Data analysis was performed by SPSS software version 22.0. The relationship between BCL11A expression level and clinical features was analyzed using the χ2 test. The independent sample t-test was used for the two groups and the one-way Analysis of Variance (ANOVA) was used among multiple groups. The overall survival (OS) and progressionfree survival (PFS) were evaluated by the Kaplan-Meier method. Statistical significance was defined as p < 0.05.

The mRNA Expression of BCL11A in Different Human Cancers
The differences in BCL11A between various tumor tissues and the matched normal tissues were analyzed using previously published microarray gene expression datasets. Results showed that the expression level of BCL11A was higher in most cancers compared with normal tissues, such as cholangiocarcinoma, colon adenocarcinoma, esophageal carcinoma, lung cancer, kidney cancer, and so on (Fig. 1A). The prognostic value of BCL11A in predicting patient outcomes was further investigated in the PrognoScan database. In the four datasets, high expression of BCL11A was related to shorter OS or disease-free survival (DFS) (Cox P value < 0.05) (Fig. 1B).
Compared with other cancers, higher expression levels of BCL11A were observed in NB cell lines and primary tumors (Fig. 1C). Moreover, the BCL11A expression level was high in normal embryonic tissues. The above results strongly implied that BCL11A might play a vital role in the onset of neuroblastoma.

Elevated Expression of BCL11A was Correlated with Lower Survival in NB Patients
To explore the correlation between BCL11A expression and clinical outcomes of NB patients, clinical data and tumor tissues were studied in 53 patients. The patients consisted of 31 boys and 22 girls. Median age at diagnosis was 28 months. Of all 53 patients, 32 cases were neuroblastoma (NB), 3 cases were Ganglioneuroblastoma nodular type (GNBn), 17 cases were Ganglioneuroblastoma intermediate (GNBi), and one case was ganglioneuroma (GN). According to the International Neuroblastoma Staging System (INSS), 9 cases were stage 1, 7 cases were stage 2, 8 cases were stage 3, 24 cases were stage 4, and 5 cases were stage 4s. Among the 53 subjects, 23 received preoperative chemotherapy.
The staining of BCL11A in NB tissues was observed in the cytoplasm and nucleus of tumor cells. As shown in Fig.  (2A), NB patients with high expression of BCL11A in tumor tissues had significantly poorer OS than those with low expression (p=0.010, Fig. 2A). Among the 53 patients, 17 (32.1%) cases developed tumor progression or recurrence and 12 of them were defined as having high expression of BCL11A. Compared with the low-expression group, patients in high expression group were more likely to present with recurrence or progression (p=0.034, Fig. 2B). Next, correlations between BCL11A and clinicopathological variables were analyzed, including gender, age, INSS stage, risk, MYCN status, pathological categories, and metastasis at diagnosis (Table 1). Interestingly, the expressions of BCL11A were closely correlated with some indicators of NB progression, such as high-risk group (P=0.018). The above results suggested that BCL11A expression had a positive correlation with undesirable clinical characteristics in NB.

BCL11A was a Potential Prognostic Factor in Patients with NB
SEQC and Asgharzadeh dataset analysis provided further evidence of the correlation between BCL11A and the surviv- al of NB patients. A total of 498 patients in the SEQC dataset and 249 patients in the Asgharzadeh dataset with complete follow-up information and gene expression profiles were recruited for survival analysis. In the SEQC dataset, patients with MYCN gene amplification and advanced stage NB were associated with a high expression of BCL11A (Fig. 2C). Kaplan-Meier survival analysis showed that patients with high expression of BCL11A had a poorer prognosis in both datasets (p 0.05, Fig. 2D, 2F). In the Asgharzadeh dataset, high expression of BCL11A was significantly correlated with unfavorable histological subtypes such as undifferentiated and poorly differentiated NB and high mitosis karyorrhexis index (MKI) (Fig. 2E). Taken together, high expression of BCL11A was related to high-risk and poor prognosis of neuroblastoma.

Estimation of Relative Expression of BCL11A in Six Cell Lines
To explore the effect of the BCL11A gene in neuroblastoma, cell culture experiments were used to provide more   convincing evidence. Using 293T cells as control, the transcription level and protein expression of BCL11A were investigated by RT-PCR and Western blot in SK-N-AS, SK-N-BE(2), IMR-32, SK-N-SH, and SH-SY5Y. Among the five NB cell lines, IMR-32 and SK-N-BE(2) possessed high expression of BCL11A in mRNA and protein levels (Fig. 3A,  B).
Transient transfections were performed in SK-N-BE(2) and IMR-32 cell lines using the procedures described above. After interfered siRNA in the two cell lines, the mRNA and protein expressions of BCL11A were significantly decreased (Fig. 3C, D).

Impaired Expression of BCL11A Inhibited Cell Proliferation
SK-N-BE(2) and IMR-32 cell growth were monitored via CCK8 assays for 3 consecutive days after siRNA transfection. Knockdown of BCL11A could significantly inhibit the proliferation in both IMR-32 and SK-N-BE(2) cell lines (Fig. 4A, B). What's more, colony formation assays demonstrated that, compared with control cells, knockdown of BCL11A remarkably suppressed the colony-forming ability (Fig. 4C, D).
To further explore the role of BCL11A on the NB cell proliferation stage, flow cytometry was performed to analyze cell cycle arrest. As shown in Figs. (4E, 4F), treatment with siRNA in SK-N-BE(2) and IMR-32 cells resulted in an increased accumulation of cell populations in the G0/G1 phase. These results indicated that BCL11A might promote NB cell proliferation.

Knockdown of BCL11A Suppressed Cell Invasion and Metastasis
Transwell invasion assay demonstrated that NB cell lines with BCL11A-knockdown remarkably reduced the invasive capacity (Fig. 5A, B). Meanwhile, the wound healing assay revealed that knockdown of BCL11A significantly inhibited cell migration in SK-N-BE(2) and IMR-32 (Fig. 5C, D). These results demonstrated that the expression of BCL11A was related to the ability of tumor migration and invasion, which may be related to tumor metastasis.

BCL11A Might Promote EMT via Regulating the PI3K/Akt Signaling Pathway
EMT is a critical process for cancer metastasis. To examine the influence of BCL11A expression on EMT in NB, the expression of epithelial and mesenchymal markers was measured by western blot. The results indicated that the expression level of the epithelial marker E-cadherin increased, whereas the mesenchymal markers such as N-cadherin, vimentin, and Slug decreased in si-BCL11A NB cells (Fig. 6A,  B). Therefore, we speculated that BCL11A regulated EMT in NB cells through EMT-associated transcription factors. According to past studies, EMT was regulated through several signaling pathways, including PI3K/AKT and Wnt/β-catenin [8,13]. As shown in Figs. (6C and 6D), the protein of phosphorylated Akt remarkably decreased in si-BCL11A NB cells, whereas there was no impact on total Akt protein expression. These results gave us a conjecture that BCL11A may induce EMT in NB cells through the PI3K/AKT signaling pathway, but more evidence was needed to confirm it.

DISCUSSION
Therefore far, mount studies have demonstrated that the overexpression of BCL11A in many tumors was correlated with poor prognosis [9,[16][17][18][19]. Consistent with these findings, our study identified that the prognosis of patients with high BCL11A expression was worse than those with low expression in neuroblastoma. Follow-up data also showed higher frequencies of recurrence or progression in tumors with high BCL11A expression. Compared with the non-highrisk group, the expression levels of BCL11A in the high-risk group were remarkably higher, either in our NB patient cohort or in the validation cohort. Although there was a previous study suggesting the possible association of BCL11A expression with high-risk neuroblastoma by database mining [11], our study further demonstrated the prognostic value of BCL11A in NB patients for the first time.    By analyzing the correlation between gene expression and clinical characteristics in patients with malignant solid tumors, the expression of BCL11A was significantly confirmed to be positively correlated with some tumor metastases, such as breast cancer [20], liver cancer [21], laryngeal squamous cell carcinoma [22], lung squamous cell carcinoma [23], and so on. Studies found that BCL11A could participate in epithelial-mesenchymal transition and promote breast cancer metastasis by the Wnt/β-catenin signaling pathway [8]. To explore the function of BCL11A in tumor growth and metastasis, functional tests were conducted by knocking down BCL11A in two neuroblastoma cell lines. The results prompted that down regulated expression of BCL11A inhibited the proliferation and invasion of neuroblastoma in vitro, which was similar to the results in NK/T lymphoma and prostate cancer [7,24]. However, in our clinical study, patients with high BCL11A expression did not have obvious distant metastasis (p=0.075). This may be due to an insufficient sample size or a short follow-up period. Because, notably, one case with high expression of BCL11A in our study presented recurrence in the fifth year after being considered clinically cured, which means that high expression of BCL11A is detrimental to long-term prognostic outcomes.
The current mechanism research in neuroblastoma was limited to the role of BCL11A in cell apoptosis [12]. This study provided new evidence of tumorigenesis of BCL11A by promoting neuroblastoma cell proliferation and metastasis. The activation of EMT is commonly reflected as a vested feature of malignancy. EMT was also reported to be associated with the migratory and invasive properties of human NB cells. Phosphatidylinositol-3-kinase (PI3K) is a key signaling molecule in many cell activities, which can regulate a series of biological processes such as cell division, differentiation, apoptosis and so on. Especially, there is increasing evidence that the PI3K/Akt pathway plays an important role in the development and progression of NB [25]. It may be considered a novel therapeutic strategy in NB [26,27]. The PI3K/Akt signaling pathway can induce the EMT process and inhibit the transcription of E-cadherin. In our study, we found that downregulation of BCL11A depressed EMT program in NB cells, including upregulation of the epithelial marker E-cadherin, as well as downregulation of mesenchymal markers (N-cadherin, slug, and vimentin). Although the process of mesenchymal to epithelial transition mediated by downregulation of BCL11A was incomplete, changes in cell migration and invasion did occur.
MYCN is a member of the MYC family of protooncogenes encoding the transcription factor N MYC. It is known for its onco-genetic role and mechanisms in the prognosis of neuroblastoma and is considered one of the prominent targets for NB therapy [28]. According to our results, the expression level of BCL11A in SK-N-BE(2) and IMR-32 was higher than in SK-N-AS, SK-N-SH and SH-SY5Y cell lines. Interestingly, it has been reported that SK N BE (2) and IMR-32 cell lines are known to have MYCN amplification, whereas the other three cell lines are not [29]. What's more, in the SEQC dataset, patients with MYCN gene amplification were associated with a high expression of BCL11A (Fig. 2C). Therefore, there might be crosstalk between BCL11A and MYCN involved pathways. This speculation requires further experimental to verify. Interestingly, the expression of BCL11A was different in gender, with higher BCL11A expression in boys with NB. It was previously reported that neuroblastoma was more common in boys than in girls, which may contribute to this discrepancy [2,30]. Further research with larger sample sizes is warranted.

CONCLUSION
In conclusion, this study clarified the high expression level of proto-oncogene BCL11A in neuroblastoma tumor tissue. Moreover, data from our case, along with data from online datasets, revealed that the high expression of BCL11A in NB patients was notably related to high-risk, distant metastasis, and poor prognosis. Functional experiments verified that BCL11A played a regulatory role in cell proliferation, invasion, and migration in NB. The potential mechanism of BCL11A in NB cells may induce EMT through the PI3K/AKT signaling pathway. Therefore, BCL11A may serve as a novel prognostic predictor and a promising target for NB therapy.

ETHICS APPROVAL AND CONSENT TO PARTICI-PATE
The ethics approval was granted by the Ethics Committee of Xinhua Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, China (approval no. XHEC-D-2021-145).

HUMAN AND ANIMAL RIGHTS
No animals were used in this study. The collection and application of specimens from patients abide by the process according to the ethical standards formulated in the Helsinki Declaration.

CONSENT FOR PUBLICATION
Written informed consent was obtained from the patient's guardian.

AVAILABILITY OF DATA AND MATERIALS
Data analyzed in this article were available in public databases (TIMER, PrognoScan, and R2). The results published here are partly based upon data generated by the Tumor Immune Estimation Resource (TIMER) (http://timer. cistrome.org/) and the R2 genomics analysis and visualization platform (http://r2.amc.nl/).